Determination of Phytochemical Constituent, Antioxidant Activity, Total Phenol and Total Flavonoid of Extract Ethanol Phyllanthus emblica Fruit

Phyllanthus emblica (PE) is a plant that is regularly found in Indonesia, particularly on Sumatra island, under the name Balakka. In India, this plant is also commonly found and widely utilised in Ayurvedic medicine under the name Indian gooseberry. PE has long been used on a regular basis to promote hair development, relieve constipation, and alleviate fever and pain. Phyllanthus emblica is a member of the Euphorbiaceae family and is found across the subtropics and tropics, including China, India, Malaysia, and Thailand. The Phyllanthus emblica fruit is quite popular due to its high vitamin C and phenolic content. According to several reports, Phyllanthus emblica fruit possesses antioxidant, immunomodulatory, and anticancer properties, Analgesic, anti-pyretic, antidiabetic, and antimicrobial.1-6


INTRODUCTION
Phyllanthus emblica (PE) is a plant that is regularly found in Indonesia, particularly on Sumatra island, under the name Balakka. In India, this plant is also commonly found and widely utilised in Ayurvedic medicine under the name Indian gooseberry. PE has long been used on a regular basis to promote hair development, relieve constipation, and alleviate fever and pain. Phyllanthus emblica is a member of the Euphorbiaceae family and is found across the subtropics and tropics, including China, India, Malaysia, and Thailand. The Phyllanthus emblica fruit is quite popular due to its high vitamin C and phenolic content. According to several reports, Phyllanthus emblica fruit possesses antioxidant, immunomodulatory, and anticancer properties, Analgesic, anti-pyretic, antidiabetic, and antimicrobial. [1][2][3][4][5][6] All components of Phyllanthus emblica have been extensively employed in a variety of traditional remedies, including Indian Medicine (Ayurveda), Chinese Traditional Medicine, Tibetan Medicine, and Greek Arabic Medicine. Minority populations of southwest China use the root of Phyllanthus emblica to treat Eczema and the fruit to treat jaundice and diarrhoea. Additionally, it is utilised as an astringent and hemostatic in Nepal. 7,8 The bark of Phyllanthus emblica has antioxidant activity and radical scavenging due to its polyphenol compounds 9,10 . In China, the bark of Phyllanthus emblica is utilised for tannin extraction due to its high tannin content (between 21 and 33 percent). Numerous pharmacological investigations have found Phyllanthus emblica but have concentrated on the fruit, with other portions, such as the bark, receiving less attention. PE is an excellent source of metabolite chemicals, which include flavonoids, saponins, tannins, steroids, and glycosides. The flavonoid compounds contained in PE are kaempferol-3-O α-L-(6″-methyl)rhamnopyranoside, kaempferol-3-O-α-L-(6″-ethyl) rhamnopyranoside, and other compounds, such as Triacontanol, Triacontanoic acid, β-Amyrin ketone, Betulonic acid, Daucosterol, Lupeol acetate, β-Amyrin-3-palmitate, Gallic acid, Betulinic acid, Ursolic acid, Oleanolic acid, Quercetin, Rutin, and Bisabolane. Also, PE fruit is rich in vitamin C, luteolin, and corilagin. [11][12][13][14] This study aim to determine the antioxidant activity, total phenol, and total flavonoid of Phyllanthus emblica ethanol extract.

Phytochemical constituent analysis by LC-HRMS
The phytochemical analysis of Phyllanthus emblica ethanol extract was performed using the TSQ Exactive (Thermo) gradient technique (LSIH, Brawijaya University) with mobile phase A (0.1 percent formic acid in water) and phase B. (0.1 percent formic acid in acetonitrile). The 501mm1.9m Hypersil GOLD Q column was analysed for 70 minutes at a flow rate of 40L/min. CompoundDiscoverersoftwarewasused in conjunctionwithmzCloudtoanalysethe data. 16

DPPH scavenging activity
The DPPH scavenging activity was determined using a slightly modified Blois technique. We dissolved up to 25 mg EEPE in 25 mL methanol and sonicated for 30 minutes (40 0C). It was then centrifuged at 1000 rpm for 10 minutes and diluted to get 6.25 g/mL, 12.5 g/mL, 25 g/mL, 50 g/mL, and 100 g/mL concentrations. Up to 20 mg DPPH was dissolved in 100 mL methanol (200 g/mL) and sonicated for 30 minutes at 40 0C, followed by centrifugation at 100 rpm for 10 minutes and dilution to reach a control concentration of 40 g/mL. The extract solution was combined with DPPH, vortexed, and left at a temperature of 270C for 30 minutes. Itwasthenquantifiedat 517 nmusing a spectrophotometers. 17 The formula is as follows: DPPH scavenging activity (IC50) = (Absorbance Control -Absorbance Sample)/Absorbance

Total flavonoid content (TFC)
A total of 10.5 mg ethanol extract of PE was diluted in methanol to a volume of 10 ml, pipetted 0.5 ml solution, and then added 1.5 ml methanol, 0.1 ml 10% aluminum chloride solution, 0.1 ml 1 M sodium acetate solution, and 2.8 ml pure water. At a wavelength of 436 nm, measurements were taken five times. The concentration of flavonoids was determined using the substitution method in the linear regression equation and expressed as the equivalent milligrams of quercetin in 1 gramme of extract. 18

Total phenol content (TPC)
The Sanchez-Rangel method was used to determine the total phenolic content using the Folin-Ciocalteu reagent. In 10 mL methanol, 10.5 mg of PE extract was dissolved. A total of 0.5 mL ethanol PE extract was vortexed for approximately 1 minute with 2.3 mL water and 0.2 mL Folin-Ciocalteu reagent, then let to stand for 5 minutes before adding 2 mL 20% sodium carbonate and allowing to stand for 70 minutes. The measurement was performed using a spectrophotometer set to 775 nm. Gallic acid was used as a standard. 19 Total phenol was calculated by multiplying GAE/g extract by five.

Phytochemical analysis of ethanol extract of PE
Determine the composition of ethanol extract of PE used LC-HRMS to analyse its phytochemical constituents. Table 1 summarises the findings.

DPPH scavenging activity
The antioxidant activity of PE used vitamin C as a comparison, the regression equation is Y = 17.548X + 2.731 with an IC50 value of vitamin C as much as 2.69 mg/dl. The antioxidant value of EEPE can be seen in Table 2.
The table 2 above shows the ability of EEPE as an antioxidant with an IC50 value of 7.626 ± 0.41 µg/dL. It shows that the antioxidant activity of the ethanol extract of Phyllanthus emblica is strong.

Total flavonoid dan phenol content
Total flavonoids from EEPE used gallic acid as a comparison. Total flavonoids contained in the extract and total phenols from EEPE used quercetin as a comparison. The regression equation for determining phenol content is y = 0.00125X + 0.0252, while the regression equation for total flavonoids is y = 0.03689X + 0.0013. The results of total flavonoids and total phenol can be seen in Table 3   Phyllanthus emblica (PE) or Indian gooseberry has been widely used as traditional and Ayurvedic medicine in India. PE is commonly spread in Southeast Asia, including Malaysia and Indonesia. A comprehensive toxicity evaluation is needed to ensure the safe use of PE. [20][21][22] The active compounds found in PE are apigenin-7-O-(6′′-butyryl-βglucopyranoside), gallic acid, and luteolin-4'-O-neohesperidoside. 23 This compound has an antioxidant role; in this study, the scavenging ability of EEPE has an IC50 value of 7.626 ± 0.41 µg/dL (Table 2).
Another study also revealed that luteolin has several cardioprotective mechanisms by means of anti-calcium overload, other luteolin functions can also reduce radical compounds (O-, H2O2, and OH-), luteolin also has anticancer activity by stimulating pathway apoptosis. 23 Moreover, Luteolin has LD 50 higher than 5000 mg/kgBW, while Gallic acid has LD 50 more than 2000 mg/kgBW. 24  Hilma Putri Lubis, MD, OG(REI)was born in Medan on October 24, 1986, is a gynecologist and an IVF clinician in Halim Fertility Center, which is the third center with the most number of IVF cycles in Indonesia. She is also a lecturer affiliated with the Department of Obstetrics & Gynecology, Faculty of Medicine, Universitas Sumatera Utara. She has authored and co-authored multiple peer-reviewed scientific papers and presented works at many national and international conferences. Her research interests include PCOS, infertility, Assisted Reproductive Technology (ART) and reproductive genetics.
Rony Abdi Syahputra, M.Farm is an active biology lecture in Department of Pharmacology, Faculty Pharmacy, Universitas Sumatera Utara, His also active in pharmacology research especially in toxicology and cardiovascular research. his active as journal reviewer and writing paper in international journal